Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Progesterone is secreted by the corpus luteum (CL) of the ovaries, placenta, and potentially the adrenal glands. Production of progesterone by the CL is supported primarily by luteinizing hormone (LH) secreted from the anterior pituitary gland. This primary source of progesterone changes during pregnancy in the mare as the fetal-placental unit begins producing progestins around days 50 to 70 (Samper J S, Pycock J F, McKinnon A O. Current therapy in equine reproduction. Philadelphia, Pa.: Saunders Elsevier, 2007; xvi, 492 s). Serum concentrations of progesterone are high, (>10 ng/ml), for about the first 150 days of gestation due to production by both primary and secondary corpora lutea (Fowden A L, Forhead A J, Ousey J C. The endocrinology of equine parturition. Experimental and Clinical Endocrinology & Diabetes 2008; 116: 393-403). Progesterone is nearly non-detectable in maternal blood after 200 days of gestation although it is present in the placenta and fetal circulation (Silver M. Placental Progestagens in the Sheep and Horse and the Changes Leading to Parturition. Experimental and Clinical Endocrinology 1994; 102: 203-211). Pregnancy is maintained in late gestation by various progestins, including 5α-DHP, 3β-5P, ββ-diol, 20α-5P and βα-diol.
Progesterone has multiple functions in the mare. Production of this hormone is required for the maintenance of pregnancy. Progesterone produced from the corpus luteum that formed after ovulation is responsible for uterine secretions and physical embryo-uterine interactions such as embryo mobility, fixation, and orientation. In addition, both estrogen and progesterone are key components to positive- and negative-feedback loops that control the release of prolactin, FSH, and LH from the anterior pituitary as well as various other reproductive hormones (McKinnon A O, Voss J L. Equine reproduction. Ames, Iowa: Blackwell Publishing, 2005; xxv, 1137 p). The LH pulse-generating system is negatively impacted by progesterone in the mare.
Progesterone is metabolized very rapidly in the mare, primarily to 5α-pregnane derivatives. Progesterone is almost completely metabolized in one passage through the GI mucosa and liver. The metabolites are primarily excreted in the urine. Metabolic clearance rates of progesterone were not significantly different in anestrous, diestrous, ovariectomized, pregnant, or lactating mares.
Only about 1-3% of progesterone is free (ie. unbound) in the blood of mares. A majority of progesterone is bound to cortisol binding globulin (CBG) and albumin carrier proteins. The proportion of progesterone bound to CBG and albumin is somewhat controversial and may be species specific. In humans, it is reported that 80% of progesterone is bound to albumin and 18% to transcortin (CBG), while the remaining 2% is free. Once steroid hormones enter into the bloodstream via endogenous secretion or exogenous administration, they immediately interact with several plasma steroid-binding proteins. Albumin is a high-abundance protein with low steroid-binding affinity and specificity whereas CBG is a low abundance protein with high steroid-binding affinity and specificity. Plasma concentrations of CBG are much lower than that of albumin but CBG plays an important role in binding and transporting biologically active steroids in the blood. Cortisol binding globulin has a single steroid binding site with which it can bind either progesterone or cortisol with high affinity. Understanding the relationship between CBG and progesterone is of extreme importance to accurately measure total progesterone concentrations.
Progesterone is essential for the maintenance of pregnancy in the mare. Among other roles, progesterone induces the endometrium to conform to invasion of microcotyledons and gaseous exchange demands of the placenta, to stimulate endometrial glands to secrete “uterine milk” for the nourishment of the embryo, and to maintain quiescence of the myometrium (Allen W R. Luteal deficiency and embryo mortality in the mare. Reprod Domest Anim 2001; 36: 121-131; Silver M. Placental Progestagens in the Sheep and Horse and the Changes Leading to Parturition. Experimental and Clinical Endocrinology 1994; 102: 203-211). The mechanism used to ensure high blood concentrations of progesterone in the pregnant mare is different than other domestic species.
There are three sources of progestins throughout pregnancy in the mare. The initial corpus luteum that formed after the ovulation that resulted in the conceptus is solely responsible for the production of progesterone until approximately day 40. The progesterone secreted from the primary corpus luteum increases until about day 8 post ovulation at which point levels slowly begin to decrease until day 28 to 30. It is thought that the 1° CL may undergo partial regression though it does not completely regress until much later in gestation, contrary to initial belief. The 1° CL regresses along with the 2° CL around 180 to 220 days of gestation. It has been suggested that CL regression occurs as a result of the loss of the luteotropin equine chorionic gonadotropin (eCG). This is most likely not the case as eCG is nearly nondetectable at 120 days of gestation, long before the regression of primary and secondary corpora lutea.
Secondary corpora lutea form around day 40 of gestation and are a supplementary source of progesterone in the pregnant mare. They contribute to a dramatic increase in concentrations of serum progesterone. The formation of 2° CL coincides with the development of endometrial cups and subsequent secretion of equine chorionic gonadotropin (eCG), which has a luteotropic role. The number of 2° CL increase until approximately 140 days; however, the quantity of 2° CL varies greatly from mare to mare. There are two types of 2° CL, those arising from ovulation and those from luteinization without ovulation. Some refer to corpora lutea resulting from ovulation as secondary and those resulting from luteinization of anovulatory follicles as accessory. It is still unknown as to the proportion that results from ovulations compared to anovulatory luteinization although formation of 2° CL from ovulation is much more likely to occur between days 40 to 70 than after day 70. A majority of 2° CL are nearly identical in physical characteristics when compared to the 1° CL. All 2° CL regress around 180 to 220 days.
Low progesterone is of great concern in the pregnant mare. There are two main classifications that exist: 1) the absence of adequate endogenous progesterone secretion due to factors that affect the function of the CL, and 2) the inability of “normal” circulating progesterone levels to maintain pregnancy. Examples of the first include accidental PGF2α administration, luteolysis resulting from endotoxemia, and failure of maternal recognition of pregnancy. Examples of the second include mares that consistently lose their pregnancy yet have normal serum progesterone levels, placentitis, and impending abortion due to stress in late gestation. Low luteal progesterone production may arise from a variety of mechanisms including primary corpus luteum insufficiency, luteolysis due to uterine inflammation (endometritis) and subsequent release of PGF2α, failure of mechanisms responsible for maternal recognition of pregnancy followed by luteolysis, luteolysis due to systemic endotoxemia, and stress. Luteal insufficiency or inadequate production of progesterone by the corpus luteum has been proposed as a cause of early pregnancy loss; however, there is very limited evidence to support primary luteal insufficiency as the true cause. In a study conducted by Irvine et al. (1990; Equine Veterinary Journal; 22: 104-106), it was found that out of 17 mares that exhibited early pregnancy loss, only one was associated with low progesterone levels in maternal circulation.
Accurate measurement of progesterone in the normal cycle is of interest to determine the phase of the estrous cycle (ie. estrus versus diestrus). This can be extremely beneficial if one is unable to ultrasound a mare or if a mare has “silent heats.” Accordingly, a need exists in the art for the rapid and accurate detection of circulating progesterone levels in the mare in order to more closely monitor and also maintain pregnancy in subject animals.